1. Field of the Invention
The present invention relates generally to the fields of molecular virology and vaccine development. More specifically, the present invention provides recombinant respiratory syncytial viruses in which the genes encoding the attachment protein G, the fusion protein F, and the Small Hydrophobic protein SH were deleted and replaced by a foreign/heterologous gene or in which the individual gene products or foreign/heterologous gene products were provided in trans.
2. Description of the Related Art
Human respiratory syncytial virus (HRSV) is a major cause of severe lower respiratory tract disease in infants and children worldwide as well as in immunosuppressed individuals and the elderly (Pringle, 1987; Couch et al., 1997; Han et al., 1999). Amidst ongoing efforts to develop human respiratory syncytial virus specific vaccines and therapeutic agents, prevention and treatment of human respiratory syncytial virus disease remain a significant challenge. Human respiratory syncytial virus is the type species of the genus Pneumovirus within the family Paramyxoviridae and contains a negative-sense, single-stranded RNA genome of 15,222 nucleotides that expresses eleven known proteins from ten genes (Huang and Wertz, 1983; Collins et al., 1984). Three proteins, SH (small hydrophobic), G (attachment), and F (fusion), have been characterized as transmembrane glycoproteins and are detected in purified virions (Anderson et al., 1992; Huang et al., 1985). On the surface of infected cells, the G and F proteins concentrate in cell-associated, virus-induced filamentous structures with variable lengths of up to 10 μm (Bächi and Howe, 1973; Fuchs and Bächi, 1975; Roberts et al., 1994; Buchholz et al., 2000; Stope et al., 2001).
The SH protein is a small integral membrane protein of unknown function, with a relatively low amino acid conservation among human respiratory syncytial virus strains (Collins and Wertz, 1985; Collins et al., 1990). Previous studies indicate that SH is dispensable for human respiratory syncytial virus growth in cell culture, and its absence has little impact on the ability of the virus to replicate in the respiratory tracts of mice and chimpanzees (Bukreyev et al., 1997; Karron et al., 1997; Whitehead et al., 1999; Techaarpornkul et al., 2001).
The G gene of human respiratory syncytial virus expresses both a type II membrane-anchored glycoprotein and a soluble protein (Wertz et al., 1985, 1989; Hendricks et al., 1987; Roberts et al., 1994). G protein is heavily O-glycosylated and shows significant structural similarities to mucinous proteins (Wertz et al., 1985, 1989). The G protein was initially characterized as providing an attachment function (Levine et al., 1987), and domains in G have since been identified that bind to sulfated glycosaminoglycans on the cell surface in vitro (Feldman et al., 1999; Martinez and Melero, 2000). The requirement for G protein in infectivity in cell culture varies depending on the cell type. Both a cold-adapted virus in which most of the sequence encoding the SH and G proteins is absent, and a n engineered virus lacking the G gene replicate efficiently in Vero cells. However, replication of these G-deleted viruses is significantly impaired in HEp-2 cells as well as in mice, cotton rats, and humans (Karron et al., 1997; Teng et al., 2001).
The fusion protein, F, is a type I transmembrane glycoprotein that mediates the formation of syncytia typically observed in human respiratory syncytial virus infected cells (Walsh and Hruska, 1983). F protein is thought to direct entry of human respiratory syncytial virus at the plasma membrane in a pH-independent manner (Srinivasakumar et al., 1991). Among the transmembrane glyoproteins, F appears to be a critical component for virus transmission, as F, matrix (M) protein, and the nucleocapsid were found to be the minimal requirements for production of infectious particles, and viruses that express F as the only glycoprotein propagated efficiently in Vero cells (Karron et al., 1997; Teng and Collins, 1998; Techaarpornkul et al., 2001; Teng et al., 2001).
The G and F proteins contain the major antigenic epitopes of human respiratory syncytial virus, and their roles in the antihuman respiratory syncytial virus virus immune response have been investigated extensively (Wertz et al., 1987; Stott et al., 1987; Murphy et al., 1990; Tripp et al., 1999; Sullender, 2000; Graham et al., 2002; Varga and Braciale, 2002). In contrast, relatively few studies have addressed the roles of SH, G, and F in viral entry, assembly, and transmission. In addition to the SH and G deletion studies described above, glycoprotein substitution studies with bovine respiratory syncytial virus (BRSV) showed that the human respiratory syncytial virus G and F proteins could functionally replace their homologs in BRSV, and that the bovine parainfluenza virus (BPIV) HN and F proteins could functionally replace the BRSV G and F proteins (Buchholz et al., 1999; Stope et al., 2001). However, to date infectious human respiratory syncytial viruses lacking an F gene from the Paramyxoviridae family have not been reported.
Current work to develop a safe and effective vaccine against respiratory syncytial virus is developing rapidly and it is now possible to recover respiratory syncytial virus from cDNA clones and to engineer viruses to specification. However, previous vaccine attempts with killed vaccine revealed that vaccination resulted in a n inappropriate immune response that led to more severe disease upon challenge. The cause of this phenomenon has not been clearly elucidated. Attempts to dissect the problem have been made with isolated viral components but not within the context of an infectious virus.
Thus, the prior art is deficient in the lack of improved recombinant respiratory syncytial viruses useful for the testing and development of vaccine for respiratory syncytial virus. The present invention fulfills this long-standing need and desire in the art.